Excessive serine from the bone marrow microenvironment impairs megakaryopoiesis and thrombopoiesis in Multiple Myeloma

Thrombocytopenia is a major complication in a subset of patients with multiple myeloma (MM). However, little is known about its development and significance during MM. Here, we show thrombocytopenia is linked to poor prognosis in MM. In addition, we identify serine, which is released from MM cells into the bone marrow microenvironment, as a key metabolic factor that suppresses megakaryopoiesis and thrombopoiesis. The impact of excessive serine on thrombocytopenia is mainly mediated through the suppression of megakaryocyte (MK) differentiation. Extrinsic serine is transported into MKs through SLC38A1 and downregulates SVIL via SAM-mediated tri-methylation of H3K9, ultimately leading to the impairment of megakaryopoiesis. Inhibition of serine utilization or treatment with TPO enhances megakaryopoiesis and thrombopoiesis and suppresses MM progression. Together, we identify serine as a key metabolic regulator of thrombocytopenia, unveil molecular mechanisms governing MM progression, and provide potential therapeutic strategies for treating MM patients by targeting thrombocytopenia.

free survival (PFS, right) in NDMM patients with normal platelet counts (120-300×10 9 /L, n = 515), high platelet counts (>300×10 9 /L, n = 61) and low platelet counts (≤120×10 9 /L, n = 291). c Kaplan-Meier analysis of OS in MM patients with normal platelet counts (120- 23). f The assessment of the correlation between platelet counts and numbers of MKs in BM (n = 114, two-sided Pearson test). g Tumorassociated luminescence intensity in live 5TGM1 mice at week 3, 4, 5 and 6 (n = 6). h Representative images of MKs in the BM from 5TGM1 mice with normal platelet counts or low platelet counts by using Hematoxylin staining. i Statistical analysis of the number of MKs per mm 2 in the BM of 5TGM1 mice with normal platelet counts (n = 4) or low platelet counts (n = 3). j Representative plot for MKs was detected in 5TGM1 mice. CD41 positive cells with high SSC are MKs. k Flow cytometry analysis of the proportion of MKs in BM from the control mice and 5TGM1 mice with different tumor burden (mean ± SD, n = 7 in ctrl group, n = 4 in group with IgG2b less than 3mg/mL, n = 4 in group with IgG2b range from 3 to 6mg/mL, n = 5 in group with IgG2b more than 6mg/mL). Results represent means ± SD. l Gating strategy for LSK (Lin − /Sca-1 + /c-Kit + ), CMPs (Lin − /Sca-1 − /c-Kit + /CD34 + /CD16/32 − ) and MEPs (Lin − /Sca-1 − /c-Kit + /CD34 − / CD16/32 − ). m Flow cytometry analysis of the proportion of LSKs, CMPs, and MEPs in the BM from the control mice and 5TGM1 mice with different tumor burden (mean ± SD, n = 15 in ctrl group, n = 11 in group with IgG2b less than 3 mg/mL, n = 6 in group with IgG2b range from 3 to 6 mg/mL, n = 7 in group with IgG2b more than 6 mg/mL). Unpaired two-sided t-test were used in d, e, i, k, m; Two-sided log-rank (Mantel-Cox) test were used in b, c. Source data are provided as a Source Data file. The ploidy of differentiated cells at day 12 treated with vehicle or serine (mean ± SD, n = 3 independent experiments). c The apoptosis rate of CD41a + CD42b + cells at day 12 of treatment with vehicle or serine (mean ± SD, n = 3 independent experiments). d The level of serine in the serum of 5TGM1 mice at week 0, 3, 6 was detected by using HPLC (mean ± SD, n = 5). e The correlation between platelet counts and serum serine level in 5TGM1 mice (n = 13). f The serum serine level in 5TGM1 mice fed with the control diet or serine-free diet, as detected with HPLC (mean ± SD, n = 5). g The proportion of LSK, CMP, MEP and MK in 5TGM1 mice fed with the control diet or serine-free diet (mean ± SD, n = 5). h Statistical analysis of the percentage of apoptotic platelets in HDderived platelets incubated with serum from MM patients with low or high serine levels (mean ± SD, nMMlow = 13, nMMhigh = 15). i, j Gating strategy for Annexin V + apoptotic platelets and statistical analysis of the percentage of apoptotic platelets in HD-derived platelets and control mouse-derived platelets incubated with vehicle, 2mM and 8mM serine. Results represent means ± SD. Unpaired two-sided ttest were used in f, g, h; One-way ANOVA followed by Dunnett's multiple comparison test were used for a, b, c, d, i, j; Twosided Pearson test was used in e. Source data are provided as a Source Data file.

Supplementary Figure 3 Serine entry into one-carbon metabolism in cells undergoing megakaryocytic differentiation. a
Schematics of the serine metabolic flux experiments. b The proportions of 13C-labeled metabolites in CD34 + cells undergoing MK differentiation (at day 6, 9 and 12) after the exposure to 13 C3-serine for 2 h. c Generation of CD41a + CD42b + cells at day 12 from CD34 + cells infected with lentivirus containing Scramble and SLC38A1-shRNA in the presence of vehicle or methionine (Met) (mean ± SD, n = 3 independent experiments). d Generation of CD41a + CD42b + PLP at day 12 from CD34 + cells infected with lentivirus containing Scramble and SLC38A1-shRNA in the presence of vehicle or Met (mean ± SD, n = 3 independent experiments). e Overview of onecarbon metabolism around the folate cycle and methionine cycle. f Assessment of mRNA level of MAT2A in differentiated cells treated with vehicle or serine at day 12 by using RT-qPCR (mean ± SD, n = 3 independent experiments). g Flow cytometry analysis of the percentage of CD41a + CD42b + MKs from CD34 + cells infected with Scramble or MAT2A-shRNA virus in the presence vehicle or serine (mean ± SD, n = 3 independent experiments). h Generation of CD41a + CD42b + PLPs at day 12 from CD34 + cells infected with Scramble or MAT2A-shRNA virus in the presence vehicle or serine (mean ± SD, n = 3 independent experiments). Unpaired two-sided t-test were used in c, d, f, g, h. Source data are provided as a Source Data file. a Relative mRNA levels of methyltransferase were detected in differentiated cells treated with vehicle or 8mM serine at day 12 by using RNA-seq (mean ± SD, n = 2 independent experiments). b Protein levels of SETDB2 and H3K9me3 were detected with immunoblotting in differentiated cells treated with vehicle or methionine at day 12. c Protein levels of SHMT2 and H3K9me3 were detected with 8 immunoblotting in differentiated cells infected with Scramble or SHMT2-shRNA at day 12. d The distribution of peaks in differentiated cells treated with vehicle or 8mM serine. e Relative mRNA levels of predicted downregulated genes were detected in differentiated cells treated with vehicle and serine by using RT-qPCR (mean ± SD, n = 3 independent experiments). f The methylation status of the CpG islands in SVIL, FRYL and SHANK3 promoters in differentiated cells treated with vehicle or serine, detected using Methylation-Specific PCR (MS-PCR). g Generation of CD41a + CD42b + cells at day 12 from CD34 + cells infected with lentivirus containing scramble and SVIL-shRNA or SHANK3-shRNA (mean ± SD, n = 3 independent experiments). h, i Phase-contrast images of proplatelet formation and generation of CD41a + CD42b + PLPs at day 12 from CD34 + cells infected with lentivirus containing scramble, SVIL-shRNA or SHANK3-shRNA (mean ± SD, n = 3 independent experiments). The scale bar is 20 µm. j Protein levels of SVIL were detected with immunoblotting in differentiated cells treated with vehicle or serine at day 12 of differentiation. k Protein levels of SVIL were detected with immunofluorescence in CD41 + cells of 5TGM1 mice fed with ctrl diet or serine-free diet. The scale bar is 20 µm. l Phase-contrast images of proplatelet formation at day 12 from CD34 + cells infected with lentivirus with EV or RUNX1 in the presence or the absence of serine.
The scale bar is 20 µm. m CD41a + CD42b + PLPs at day 12 from CD34 + cells infected with lentivirus with EV or RUNX1 in the presence or the absence of serine (mean ± SD, n = 3 independent experiments). n Protein levels of SETDB2 and H3K9me3 were detected with immunoblotting in 293T cells infected with lentivirus containing scramble or SETDB2-shRNA. o The binding of H3K9me3 to the promoters of the SVIL in 293T cells infected with lentivirus with scramble or SETDB2-shRNA in the presence or the absence of serine, as revealed with ChIP-qPCR (mean ± SD, n = 3 independent experiments). Each experiment was repeated independently for 3 times in b, c, f, j, k, n. Unpaired two-sided t-test were used in e, m, o; One-way ANOVA followed by Dunnett's multiple comparison test were used for g, i. Source data are provided as a Source Data file. h The level of serine in CM from ARP1-Scramble cells and ARP1-PHGDH-shRNA cells, as determined with HPLC (mean ± SD, n = 3 independent experiments). i Flow cytometry analysis of the percentage of CD41a + CD42b + MKs from CD34 + cells treated with ARP1-PHGDH-sh1 CM and ARP1-PHGDH-sh2 CM (mean ± SD, n = 3 independent experiments). j, k Representative images of proplatelet formation and generation of CD41a + CD42b + PLPs at day 12 from CD34 + cells treated with ARP1-PHGDH-sh1CM and ARP1-PHGDH-sh2 CM (mean ± SD, n = 3 independent experiments). The scale bar is 20 µm. l The expression profile of serine metabolism-related genes in differentiated cells at day 12 cultured with the control medium or ARP1 CM. Unpaired two-sided t-test were used in b, f, g, h, i, k.
Two-sided Pearson test was used in c. Source data are provided as a Source Data file.

Supplementary Tables
Supplementary Table 1